Synergistic effects of boron and saponin in mitigating salinity stress to enhance sweet potato growth

Salinity stress significantly hinders plant growth by disrupting osmotic balance and inhibiting nutrient uptake, leading to reduced biomass and stunted development. Using saponin (SAP) and boron (B) can effectively overcome this issue. Boron decreases salinity stress by stabilizing cell walls and membranes, regulating ion balance, activating antioxidant enzymes, and enhancing water uptake. SAP are bioactive compounds that have the potential to alleviate salinity stress by improving nutrient uptake, modulating plant hormone levels, promoting root growth, and stimulating antioxidant activity. That’s why the current study was planned to use a combination of SAP and boron as amendments to mitigate salinity stress in sweet potatoes. Four levels of SAP (0%, 0.1%, 0.15%, and 0.20%) and B (control, 5, 10, and 20 mg/L B) were applied in 4 replications following a completely randomized design. Results illustrated that 0.15% SAP with 20 mg/L B caused significant enhancement in sweet potato vine length (13.12%), vine weight (12.86%), root weight (8.31%), over control under salinity stress. A significant improvement in sweet potato chlorophyll a (9.84%), chlorophyll b (20.20%), total chlorophyll (13.94%), photosynthetic rate (17.69%), transpiration rate (16.03%), and stomatal conductance (17.59%) contrast to control under salinity stress prove the effectiveness of 0.15% SAP + 20 mg/L B treatment. In conclusion, 0.15% SAP + 20 mg/L B is recommended to mitigate salinity stress in sweet potatoes.


Free proline, chlorophyll contents and carotenoids
Initially, healthy fresh leaves from the plant were collected after 50 days of tuber.Carefully, leaves were removed by hand to minimize the chances of minimal physical damage.The collected leaves were stored in a liquid nitrogen container and then transferred to the laboratory to prevent contamination and preserve their integrity.The extraction and analysis of free proline from leaf tissues were then performed following the protocol described by 22 .The absorbance was taken for the complex, made of ninhydrin and proline at 520 nm wavelength, for the final determination of free proline.For chlorophyll and carotenoids, 10 mL of 80% acetone was added to each tube, and extraction was done by taking 1 g of fresh leaf tissues in darkness at room temperature.

Chlorophyll fluorescence
The fluorescence emitted from the upper leaf surface (adaxial) was evaluated using a fluorescence monitoring system operating in the pulse amplitude modulation mode.This assessment followed the methodology as detailed by 24 .Furthermore, the photon yield of PSII (˚PSII) during illumination was computed as °PSII = (Fm − F)/Fm after 45 s of continuous light exposure.This duration ensured the attainment of a steady state for accurate measurement 25 .

Net photosynthetic rate, stomatal conductance, and transpiration rate
The net photosynthetic rate, stomatal conductance, and transpiration rate were assessed using a Portable Photosynthesis System incorporating an infrared gas Analyzer 26 .

Assay of DPPH radical scavenging activity
A 0.1 ml portion of the diluted sample was mixed with 3.9 ml DPPH solution to initiate the reaction process.The UV-spectrophotometer's absorbance was measured at 515 nm at one-minute intervals for 180 min.Consequently, a standardized 3 h reaction time was employed for all DPPH assays 27 .

Assay of ABTS radical scavenging and MDA activity
The method for evaluating ABTS radical-scavenging activity in the hydrophilic fractions followed the protocol outlined by 28 .An ABTS+ solution was prepared by mixing 8 mM of ABTS salt with 3 mM of potassium persulfate in 25 ml of distilled water.This mixture was left in darkness at room temperature for 16 h.To achieve an absorbance between 0.8 and 0.9 at 734 nm, the ABTS+ solution was diluted with 95% ethanol (approximately 600 μl ABTS in 40 ml 95% ethanol).Using a UV-spectrophotometer, absorbance at 734 nm was recorded every minute for 30 min.For measuring malondialdehyde (MDA), a marker of lipid peroxidation, the sample extract was treated with thiobarbituric acid (TBA) to generate a visible complex.The absorbance of this complex was quantified at 532 nm wavelength to determine the concentration of MDA.

Statistical analysis
Traditional statistical methods were utilized for data analysis, encompassing a two-way ANOVA to assess treatment significance.Paired comparisons underwent the Tukey test, setting significance at p ≤ 0.05.OriginPro software 29 generated cluster plots with convex hulls, hierarchical cluster plots, and Pearson correlations.

Ethics approval and consent to participate
We all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue.So, it is not applicable.Study protocol must comply with relevant institutional, national, and international guidelines and legislation.Our experiment follows the with relevant institutional, national, and international guidelines and legislation.

Vine length, vine weight, and number of leaves
For 0% SAP, the addition of 5 mg/L boron (B) resulted in 1.72% increase, while 10 mg/L B and 20 mg/L B showed more significant increases of 8.68% and 16.58%, respectively, compared to control.Moving to 0.1% SAP, the vine length was increased by 7.09%, 14.26%, and 19.91% at 5, 10, and 20 mg/L B over control.At 0.15% SAP, the vine length was enhanced by 4.93%, 9.94%, and 13.12% where 5, 10, and 20 mg/L B were applied over control.
In the case of 0% SAP, adding 5 mg/L B resulted in a 5.85% increase, 10 mg/L B led to a 13.43%, and 20 mg/L B showed a 19.56% increase in storage root yield over the control.With 0.1% SAP, adding 5, 10, and 20 mg/L B resulted in a 9.66%, 20.00%, and 27.91% increase in storage root yield compared to the control.Moving to 0.15% SAP, 5 mg/L B showed a 3.52% increase in storage root yield, 10, and 20 mg/L B exhibit 7.77%, and 10.18% increase above the control.With 0.20% SAP, applying 5, 10, and 20 mg/L B resulted in an 8.23%, 17.75%, and 28.13% rise in storage root yield from the control (Fig. 2).

Chlorophyll and carotenoid content
Under 0% SAP, adding 5, 10, and 20 mg/L B resulted in a 23.65%, 37.24%, and 53.74% increase in chlorophyll a than the control.In the 0.1% SAP, the chlorophyll a content increased by 5.11% for 5 mg/L B, 11.96% for 10 mg/L B, and 17.73% for 20 mg/L B over the control.Moving to the 0.15% SAP, 2.21%, 5.51%, and 9.84% rise in chlorophyll a was observed with 5, 10, and 20 mg/L B compared to the control.With 0.20% SAP, the chlorophyll a content showed 10.58% increase with 5 mg/L B, 18.25% with 10 mg/L B, and 22.77% with 20 mg/L B from the control (Fig. 3).
At 0% SAP, the introduction of 5 mg/L B showed a 17.64% increase in chlorophyll b, and 10 and 20 mg/L B showed a 34.29% and 59.78% increase than the control.Moving to the 0.1% SAP, the chlorophyll b content increased by 12.85% with 5 mg/L B, 25.80%, and 42.27% increase with 10 mg/L B and 20 mg/L B, respectively, then the control.At 0.15% SAP, the chlorophyll b content showed a 6.86% rise with 5 mg/L B more than the control, 12.48% with 10 mg/L B, and a 20.20% increase with 20 mg/L B. At 0.20% SAP, chlorophyll b content increased by 12.63%, 30.49%, and 45.38% with 5, 10, and 20 mg/L B over the control (Fig. 3).
Adding 5, 10, and 20 mg/L B led to a 22.04%, 36.45%, and 55.35% increase compared to the control under 0% SAP.With 0.1% SAP, applying 5 mg/L B resulted in a 7.74% increase in total chlorophyll content, 10 and 20 mg/L B led to a 16.67% and 26.08% increase than control.Adding 5 mg/L B showed a 4.06% increase, 10 mg/L B showed an 8.27% increase, and 20 mg/L B showed a 13.94% increase over control under 0.15% SAP.With 0.20% SAP, adding 5, 10, and 20 mg/L B resulted in an 11.15%, 21.69%, and 29.11% increase in total chlorophyll content compared to control (Fig. 3).
Under 0% SAP, Fv/Fm ratio showed a 3.22%, 5.25%, and 6.86% increase, and with 0.1% SAP resulted in a 4.13%, 9.63%, and 12.64% increase more than the control.Adding 5 mg/L B with 0.15% SAP led to a 2.15% increase in Fv/Fm, 10 mg/L B resulted in a 6.28% increase, and 20 mg/L B showed a 12.25% increase from the control.With 0.20% SAP, adding 5 mg/L B resulted in a 3.32% increase in Fv/Fm, 10 mg/L B led to a 7.14% increase, and 20 mg/L B showed a 9.29% increase over the control (Fig. 4).
At 0% SAP, adding 5 mg/L B resulted in a 1.95% decrease in MDA activity, while 10 mg/L B and 20 mg/L B led to 3.78% and 7.59% decrease, respectively, then the control.For 0.1% SAP concentration, adding boron at 5, 10, and 20 mg/L B led to a 4.42%, 10.07%, and 13.36% decrease in MDA activity over the control.Compared to the control, adding 0.15% SAP with 5, 10, and 20 mg/L B resulted in a 5.51%, 10.30%, and 16.44% decrease in MDA activity.With 0.20% SAP, adding 5, 10, and 20 mg/L B showed a 3.29%, 5.74%, and 11.76% decrease in MDA activity compared to the control (Fig. 6).

Convex hull and hierarchical cluster analysis
The convex hull analysis was conducted on a dataset of coordinates represented in a two-dimensional space (PC 1 and PC 2) and labeled according to different percentages of SAP (SAP).The analysis indicates that at 98.81% accuracy along PC 1 and 0.75% accuracy along PC 2, the data points within the categories of 0% SAP, 0.1% SAP, 0.15% SAP, and 0.20% SAP form distinct convex shapes or boundaries when plotted on a graph (Fig. 7A).7B).
The first cluster amalgamates variables associated with physiological processes, including photosynthetic and transpiration rates, leaf area, and chlorophyll content, suggesting an interconnectedness in plant function.Another cluster identifies variables linked to antioxidant capabilities, such as DPPH and ABTS measurements, implying a shared characteristic of the plant's antioxidative properties.Moreover, a distinct cluster forms around pigmentation-related variables like chlorophylls, carotenoids, and photon yield of PSII, potentially indicating a cohesive group related to plant pigmentation and photosynthetic activity.Additionally, the analysis reveals a cluster involving variables like MDA and proline content, likely associated with stress responses or cellular protection mechanisms within the plant (Fig. 7C).

Pearson correlation analysis
Numerous strong positive correlations are evident among multiple parameters, including vine length, vine weight, number of leaves, leaf area, root weight, chlorophyll concentrations, photosynthetic rate, stomatal conductance, and soluble protein.These correlations, approaching or reaching values close to 1, imply a direct relationship or proportionality between these factors, suggesting that changes or variations in one parameter are likely reflected in others within this group.Additionally, moderate to high positive correlations exist among variables like transpiration rate, Fv/Fm, and photon yield of PSII, indicating their interconnectedness and potential mutual influence.Conversely, strong negative correlations are observed among measures such as MDA, proline content, www.nature.com/scientificreports/DPPH, and ABTS, signaling an inverse relationship or potential opposing impacts between these variables and the rest of the parameters (Fig. 8).

Discussion
Boron is an essential micronutrient for plant growth, influencing various physiological processes.Boron is a vital micronutrient for plant growth, influencing cell wall formation, carbohydrate metabolism, nucleic acid synthesis, cell membrane stabilization, cell division, and elongation, enhancing plant development 30 .This micronutrient often acts as a cofactor for various enzymes involved in cell wall synthesis, thereby influencing elongation processes and contributing to increased vine length 31 , as shown in (Fig. 1A).Moreover, boron plays a role in sugar transport within the plant, impacting sink-source relationships.This involvement in sugar translocation can potentially contribute to increased vine weight, as shown in (Fig. 1B), as sugars are essential to produce biomass and storage compounds 32 .SAP are bio-stimulants that enhance plant growth by improving nutrient uptake, root growth, and stress tolerance 33 .They facilitate soil nutrient absorption, including boron, potentially increasing vine growth parameters.When combined with boron, SAP enhances boron's impact on cellular processes 34 .This combined effect may stimulate cell division, elongation, and metabolic activities, leading to increased vine length and leaf number, as shown in (Fig. 1A,C).The interaction between SAP and boron may trigger growthrelated gene expression, activating specific genes and improving vine growth metrics.Leaf area expansion likely results from the combined impact of SAP, known for altering cell membrane permeability and aiding nutrient uptake, and boron, crucial for cell wall formation 35 .Their collaboration enhances cell division and expansion, contributing to increased leaf area.In root development, SAP's influence on root exudation and microbial communities and boron's role in cell wall structure likely synergize to promote root growth and branching 36 .This interaction improves nutrient uptake efficiency and structural development.Additionally, the combined effect of SAP and boron on storage root yield may arise from enhanced photosynthetic efficiency due to SAP and optimized carbohydrate translocation facilitated by boron.This collaborative influence optimizes assimilate allocation, boosting storage root yield shown in (Fig. 2C).The mechanisms involve improved nutrient uptake, fortified cell walls, efficient nutrient translocation, and heightened photosynthetic efficiency.SAP may act as signaling molecules affecting growth and stress response pathways, while boron supports enzymatic reactions and structural components crucial for these processes 37 .The increase in chlorophyll levels, particularly chlorophyll a and b, owes credit to boron's pivotal role in chlorophyll synthesis 38 .Boron aids chlorophyll formation by supporting chloroplast membrane stability, which is crucial for assembling photosystems and electron transport in photosynthesis 39 .It also influences enzymes involved in chlorophyll biosynthesis, enhancing precursor conversion into chlorophyll.The dose-dependent response to boron levels underscores its importance in optimizing chlorophyll production.Furthermore, boron's impact on chloroplasts indirectly affects carotenoid accumulation, which is pivotal as a protective pigment in photosystems 40 .Boron's positive influence on chlorophyll likely fosters an environment for increased carotenoid synthesis, mutually enhancing light capture and energy utilization in photosynthesis 41 .The interplay between SAP and boron observed in this study might involve SAP aiding boron uptake or influencing plant processes, indirectly shaping boron's availability or metabolic effects within plant tissues.Boron's pivotal role in photosynthesis involves optimizing enzymatic reactions within chloroplasts, enhancing chlorophyll synthesis, and regulating carbon fixation processes, which substantially increases photosynthetic rates 42 .Its influence extends to stomatal regulation, impacting water movement and nutrient transport, which are crucial for overall plant growth.SAP complement these effects by potentially improving membrane integrity and nutrient uptake 43 .The rise in Fv/Fm ratios signifies improved photosystem efficiency, attributed to boron's optimization of electron transport and chlorophyll protection, with SAP potentially enhancing these benefits.Boron influence on PSII stability contributes to increased photon yield, potentially supported by SAP' role in PSII protection against oxidative stress 44 .The fluctuations in soluble protein levels may arise from intricate interactions among boron, SAP, and protein metabolism pathways, influencing protein synthesis and turnover processes.Boron supplementation and SAP presence affect plant growth, stress responses, and proline content.Boron decreases proline content, suggesting osmotic balance regulation and reducing proline accumulation in response to stress 45 .According to the hierarchical cluster analysis, MDA is the least parameter to increase plant growth, as shown in (Fig. 7C).The decrease in MDA, a marker of lipid peroxidation and oxidative stress, with boron addition hints at its role in mitigating oxidative damage.Boron might participate in antioxidant enzymatic systems or directly scavenge reactive oxygen species (ROS), reducing lipid peroxidation and oxidative stress levels 46 .This could signify a protective mechanism against oxidative damage induced by stressors.Regarding growth enhancement mechanisms, the observed alterations in proline content, MDA activity, and antioxidant capacities could indirectly contribute to improved growth.Reduced proline levels may signify an optimized stress response, allowing plants to allocate resources toward growth rather than stress adaptation.Decreased lipid peroxidation and enhanced antioxidant activities might contribute to a less stressful cellular environment, enabling plants to allocate energy and resources toward growth processes.

Conclusion
In conclusion, the combination of 0.15% SAP and 20 mg/L B emerged as the most effective treatment in mitigating salinity stress for sweet potatoes.This combination notably improved various growth parameters and physiological aspects, highlighting its potential as a recommended amendment for addressing salinity-related issues in this crop.The study suggests a potential solution to alleviate salinity-induced limitations on sweet potato cultivation, potentially revolutionizing crop management strategies and contributing to food security.Future research should explore this treatment's long-term effects and scalability under different environmental conditions.

Figure 1 .
Figure 1.Effect of different concentrations of saponin (SAP) and boron (B) treatments on vine length, vine weight, and number of leaves of sweet potato.Each bar is an average of n = 4 having ± SD showing significant changes at p ≤ 0.05 by applying the Tukey test.

Figure 2 .
Figure 2. Effect of different concentrations of saponin (SAP) and boron (B) treatments on leaf area, root weight, and storage root yield of sweet potato.Each bar is an average of n = 4 having ± SD showing significant changes at p ≤ 0.05 by applying the Tukey test.

Figure 3 .
Figure 3.Effect of different concentrations of saponin (SAP) and boron (B) treatments on chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids of sweet potato.Each bar is an average of n = 4 having ± SD showing significant changes at p ≤ 0.05 by applying the Tukey test.

Figure 4 .
Figure 4. Effect of different concentrations of saponin (SAP) and boron (B) treatments on photosynthetic rate, stomatal conductance, transpiration rate, and Fv/Fm of sweet potato.Each bar is an average of n = 4 having ± SD showing significant changes at p ≤ 0.05 by applying the Tukey test.

Figure 5 .
Figure 5.Effect of different saponin (SAP) and boron (B) treatment concentrations on sweet potato photon yield of PSII and total soluble protein.Each bar is an average of n = 4 having ± SD showing significant changes at p ≤ 0.05 by applying the Tukey test.

Figure 6 .
Figure 6.Effect of different concentrations of saponin (SAP) and boron (B) treatments on proline content, malondialdehyde (MDA), DPPH, and ABTS of sweet potato.Each bar is an average of n = 4 having ± SD showing significant changes at p ≤ 0.05 by applying the Tukey test.

Table 1 .
Pre-experimental soil and irrigation characteristics.